P401: NEXT-GENERATION FLOW CYTOMETRY TO DETECT MEASURABLE RESIDUAL DISEASE IN ACUTE MYELOID LEUKEMIA USING A 19-COLOR FULL SPECTRUM FLOW CYTOMETRY PANEL
Authors: H. Fokken, N. Kattre, A. Kloos, M. Heuser, T. Kacprowski, M. Tobias, A. Schwarzer
Journal: HemaSphere (2022)

Background: The detection of residual leukemic cells (“measurable residual disease”/MRD) has dramatic prognostic impact on relapse incidence and patient survival in AML and has become key to guide therapeutic decision making. MRD can be measured by PCR, next-generation sequencing, and conventional multicolor flow cytometry (Flow-MRD, 8-10 colors), respectively. Flow-MRD relies on the aberrant expression of surface marker combinations on leukemic blasts (leukemia-associated immunophenotypes - LAIPs), which are not found on normal hematopoietic cells. Hence, the challenge of Flow-MRD is to detect and track small populations of leukemic cells against the complex and heterogenous background of human bone marrow cells. With the development of next-generation Spectral Flow Cytometers, it is now possible to stain cells with 20-30 colors at once which would greatly increase the number of LAIPs that can be tracked and increase sensitivity of Flow-MRD when the number of available cells is limited. We hypothesized that spectral Flow-MRD allows for monitoring leukemic disease burden with unprecedented resolution. Aims: To establish a one-tube full spectrum MRD detection assay and a semi-automated computational workflow for improved detection of minimal residual disease in AML. Methods: We established a 19-color full spectrum flow cytometry panel with one tube containing CD45, CD34, CD117, CD13, CD33, HLA-DR, CD2, CD4, CD5, CD7, CD11b, CD14, CD15, CD19, CD22, CD38, CD56, CD64 and CD133. Limit of detection and quantification, linearity, intra-/inter-assay precision, and robustness were determined via spike-in assays. We measured normal bone marrow of healthy donors and MRD samples from AML patients. Data were analyzed with GemStone using its unique highly automated approach to characterizing normal cells in bone marrow based on a template model designed specifically for data generated with this panel. Results were then explored with the Cen-Se dimensionality reduction algorithm (Bagwell et al. J Biom Biostat (2019)) and subsequently compared to expert gating of data generated on the same samples using a well-established 5-Tube conventional flow cytometry panel. Results: We defined reference values for known leukemia-associated immunophenotypes using normal bone marrow. Expert analysis of data simultaneously analyzed with the full-spectrum panel and the 5-tube conventional flow cytometry panel yielded highly correlating results for the proportions of granulocytes, lymphocytes, monocytes, and immature blasts in measured bone marrow with r2 values greater than 0.90. In addition, quantitative and qualitative MRD results correlated very well when comparing semi-automated analysis of our full-spectrum panel using GemStone and expert analysis of the 5-tube conventional flow cytometry panel. Summary/Conclusion: We have developed an integrated pipeline for full-spectrum flow cytometry that improves the applicability and performance of flow-cytometry-based MRD detection in AML.